RNA interference (RNAi), a specific gene silencing method mediated by an intracellular enzyme complex using a dsRNA template, is of great current interest for drug discovery due to important advantages over older antisense methods. These advantages of include: (i) use of shorter RNA avoiding interferon induction, (ii) use of dsRNA which is more stable, and (iii) RNAi-mediated gene silencing is catalytic, so that one molecule can induce cleavage of many target mRNAs. Recent reports have touted the utility of RNAi for basic and applied research aimed at determining functions of gene products and validating drug targets. However, because nucleic acids do not readily pass through intact living cell membranes, robust techniques to deliver reagents into target cells are needed to fully realize the potential of RNAi-mediated gene silencing. We have developed a novel prototype system (called LEAP(TM)) for laser-based cell analysis and manipulation. LEAP has been used for optoinjection of reagents into target cells, most recently for achieving RNAi and subsequent high-throughput imaging to measure the RNAi effect on many individual cells. Phase I proposes to: i) evaluate new reagents for achieving RNAi with optoinjection, ii) optimize optoinjection parameters for high cell viability and high efficiency RNAi, and iii) extend results to cells and genes with high drug discovery importance. This work will demonstrate the broad utility of LEAP for in situ gene silencing by optoinjection of RNAi reagents. Our preliminary data has attracted the attention of several major pharmaceutical/biotechnology companies, validating the commercial potential of the approach described. Phase II would then propose to develop specific applications such as: i) high-throughput gene function evaluation (e.g., a different gene in each well of a 1536-well plate), ii) simultaneous silencing of separate genes in the same cells, iii) silencing whole pathways of many genes, and iv) focusing on specific cellular processes that are known to be rich in candidate drug targets. With such data from Phase II, a Phase III commercialization effort would be enabled for drug development based on LEAP and RNAi-mediated gene silencing.